Comparison of the roles of neurohormones in the regulation of blood distribution from the hearts of American and Japanese lobsters

The decapod cardiovascular system consists of a single ventricle that pumps blood into seven arteries; previous work has shown that the outflow distribution patterns of intact animals are variable. In the present study, flow recordings were made from pairs of arteries in semi-isolated hearts whilst different cardioactive hormones were infused into the heart. Each hormone (5-hydroxytryptamine, octopamine, dopamine, proctolin and F1) changed the outflow pattern, heart rate and ventricular pressure in a unique way. The probable sites of hormone action are the cardioarterial valves located at the origin of each artery except one, the dorsal abdominal. Outflow from the dorsal abdominal is controlled downstream by valves located at the origin of the segmental lateral arteries. The responses to a particular hormone were sometimes different between the hearts of American and Japanese lobsters. Accepted: 11 May 1998     

Functional assignment to JEV proteins using SVM

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).     

Chloroplast DNA phylogeography of Fagus crenata (Fagaceae) in Japan

 CpDNA variation in Japanese beech, Fagus crenata (Fagaceae), was studied in 45 populations distributed throughout the species' range. Two cpDNA regions were sequenced: the non-coding region between the trnL (UAA) 5′exon and trnF (GAA), and the trnK region (including matK). Thirteen distinct cpDNA haplotypes were recognized and each haplotype was found to be geographically structured. Two major clades (I and II+III) were revealed in phylogenetic analyses among the haplotypes using F. sylvatica as an outgroup. The haplotypes of Clade I were distributed mainly along the Japan Sea side of the Japanese Archipelago, while those of Clade II+III occurred chiefly along the Pacific Ocean side. Consequently, the distribution of the two major cpDNA clades suggests that there were two migration routes in the history of F. crenata; one along the Japan Sea and the other along the Pacific Ocean side of the Japanese Islands. Received March 19, 2001 Accepted November 22, 2001     

Rearing temperature influences flavivirus vector competence of mosquitoes

Culex annulirostris Skuse mosquitoes (Brisbane strain) were reared at 20 degrees C or 27 degrees C and the adult females were experimentally infected by feeding Murray Valley encephalitis virus (MVE). They were then maintained (a) in the insectary at 20 degrees C, after rearing at either 20 degrees C or 27 degrees C; (b) at ambient outdoor temperatures, range 12.2-28.9 degrees C, mean 19.6 degrees C; or (c) at 27 degrees C after rearing at 27 degrees C. There was no significant difference in rates of MVE infection or transmission when mosquitoes were reared and maintained constantly at 20 degrees C or 27 degrees C. However, for females kept at reduced temperature (i.e. mean = 19.6 degrees C or 20 degrees C after rearing at 27 degrees C), the infection and transmission rates of MVE were significantly reduced (2 x 8 replicates). This investigation illustrates that vector competence is depressed by decreasing temperatures for adult mosquitoes compared with those they experienced during development. Similar patterns were evident with previously published work on Japanese and St Louis encephalitis, dengue and yellow fever. The process appears to be reversible, i.e. increased temperature raises virus infection and transmission rates. It is concluded that, without incubation at warmer temperatures, flavivirus recovery from overwintering mosquitoes will be negatively biased.     

Transmissibility of Bacterial Endosymbionts Between Isolates of Acanthamoeba Spp.

ABSTRACT. Experimental transmission of two bacterial endosymbionts to symbiont-free isolates of Acanthamoeba spp. was studied to determine specificity of the host-symbiont relationship. Both symbionts originated from amoebic isolates displaying an identical mitochondrial DNA Eco RI fingerprint (group AcUW II). Symbioses were readily established in one amoebic isolate which displayed a homologous mtDNA fingerprint (group AcUW II). Exposure of a heterologous amoebic isolate (group AcUW IV) to the two symbionts resulted in either cell death or encystation without the establishment of symbioses. While symbioses were established with an amoebic isolate from a second heterologous group (AcUW I), a unique membranous sheath appeared and persisted around one of the symbionts which did not exist in the original host. An isolate representing a third heterologous amoebic group (AcUW VI) was variable in its susceptibility with one symbiont unable to infect the host and the other becoming established only after an initial reaction in which trophozoites rounded-up and floated off the substrate. These studies suggest that a specific recognition system exists between particular isolates of Acanthamoeba and their symbionts, and that the appearance of a killer phenotype is related to contact between mismatched, though recognized, pairs.     

Dual Appearances of Pigment Cells from In Vitro Cultured Embryonic Cells of Japanese Flounder: An Implication for a Differentiation–Associated Clock

The cells dissociated from developing embryos of Japanese flounder (Paralichthys olivaceus) are cultured in vitro to examine the developmental fate of their pigment cells in relation to establishment of bilaterally asymmetric integumental coloration in vivo. When neurula embryos are dissociated using trypsin–EDTA in Dulbecco's modified Ca²⁺–, Mg²⁺–free phosphate buffered saline and then cultured in vitro using L–15–based fetal calf serum–supplemented growth medium at 20°C, numerous pigment cells appear twice in the same culture with an interval of approximately 1 month even under similar culture conditions. The first group of pigment cells, which is relatively larger in cell size (about 70 μm wide) and lower in cell density, emerges within 12 hr after plating, whereas the second, which is far smaller in cell size (about 30 μm) and overwhelmingly higher in cell density than the first, does so about 1 month after plating. The timing of their appearances in vitro is in good accordance, respectively, with that observed for the larvae under normal development in vivo; the first group appears at the period corresponding to hatching, whereas the second at the period corresponding to the completion of metamorphosis. Light microscopic examinations disclose that each group of pigment cells is composed of black melanophores and reflecting leucophores, and that the population density of melanophores and leucophores in the first group at the climax of appearance is approximated as 1:4. Typical xanthophores that are distributed in the skin of the larvae of this species are scarcely observed in culture in vitro. Because of their dual synchronous appearances with about 1 month interval under the similar culture conditions, and because of their low proliferative activity during the periods from the first appearance to the second, it is presumed that both groups of pigment cells are installed with a clock set differently for their differentiation. Light and electron microscopic immunocytochemistry on cultured cells using the HNK–I antibody, which marks avian migratory neural crest cells, both disclose that the antibody cross–reacts with all these pigment cells, and that a certain number of immunoreactive unpigmented cells exist even at the time of the second appearance of pigment cells. These findings would imply that the second group of pigment cells served in a form of undifferentiated propigment cells up to metamorphosis, at which they start to differentiate under control of a clock presumably set during neurulation.     

Vascular permeability to proteins and peptides in the mouse pineal gland

Summary The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides was demonstrated by means of ultrastructural tracers. Horseradish peroxidase (HRP) and microperoxidase (MP) were injected intravenously and allowed to circulate for approximately 30 s, 1 min, 5 min, 1 or 2h. The tissue was then fixed by vascular perfusion or by immersion with aldehydes. In all experiments a pronounced extravasation of HRP and MP occurred. Transendothelial vesicular transport seemed to have occurred across the fenestrated capillaries. The most pronounced tracer labeling of vesicles was found after 1 min of MP- or HRP-circulation. The vesicles were uncoated and more than 70 % of the HRP-and MP-containing vesicles exhibited diameters between 50 and 110 nm. Furthermore, three other transcapillary pathways taken by the tracers are suggested: 1) via intercellular junctions, 2) through fenestrae and 3) via channels formed by fusion of vesicles with the luminal and abluminal cell membranes. Based on these results, it is assumed that the capillaries in the mouse pineal gland are also permeable to peptides synthesized and secreted by the pineal gland.Part of this study was presented at the EMCELL-76 meeting, Copenhagen, 1976     

Serologic Analyses of Cell-Surface Antigens of Acanthamoeba spp. with Plasma Membrane Antisera*†

SYNOPSIS. Antisera were raised against plasma membrane-enriched fractions of the species Acanthamoeba castellanii and Acanthamoeba culbertsoni to determine whether cell-surface antigens would facilitate species identification of Acanthamoeba isolated from the environment or in human infections. Acanthamoeba castellanii and A. culbertsoni plasma membranes were purified, after homogenization, by differential and isopycnic centrifugation. Electron microscopic examination of purified membrane samples showed an enrichment of membranes with a typical trilaminar structure. Occasionally, mitochondria were recognized in the electron microscope preparations. 5′-Nucleotidase, Mg²⁺-ATPase, and alkaline phosphatase were enriched 11-fold, 2-fold, and 7-fold, respectively, in the A. castellanii membranes, as determined from analyses of the enzyme activities in whole cell homogenates and membrane preparations. 5′-Nucleotidase was not detected in A. culbertsoni, but the activities of Mg²⁺-ATPase and alkaline phosphatase were increased 2- to 3-fold. Both membrane preparations showed no glucose-6-phosphatase activity and less than 5% contamination with succinic dehydrogenase. From assays of acid phosphatase activity, the most apparent contamination of the plasma membrane preparations was with membranes of phagocytic vacuoles. Acanthamoeba castellanii membrane antisera produced significant agglutination and fluorescence of homologous cells to titers of 1:8192 and 1:1024, respectively. Acanthamoeba polyphaga and Acanthamoeba rhysodes gave the most cross-reactions in heterologous tests. They were agglutinated to a titer of 1:128 and positively fluoresced to titers of 1:32 and 1:64, respectively. Antisera of A. culbertsoni membrane agglutinated homologous cells at a dilution up to 1:4096 and produced homologous fluorescent titers up to 1:512. Other than agglutination of A. polyphaga to a titer of 1:128, these antisera did not cross-react significantly with any remaining heterologous species. Three new isolates were identified with these plasma membrane antisera: 2 of them, contaminants from tumor tissue cultures, were identified as A. culbertsoni. Preliminary information is also given on the use of the membrane antisera for species identification of Acanthamoeba in several new cases of amebic encephalitis.     

Vector competence of Australian Culex gelidus Theobald (Diptera: Culicidae) for endemic and exotic arboviruses

The recent recognition of established populations of the mosquito, Culex gelidus Theobald, in Australia has raised concerns about local transmission of arboviruses. The vector competence of a mainland population of Cx. gelidus was investigated for two local alphaviruses, Ross River (RRV) and Barmah Forest (BFV) viruses, and three flaviviruses, Japanese encephalitis (JEV), Kunjin (KUNV) and Murray Valley encephalitis (MVEV) viruses. Colonised mosquitoes were exposed to virus via blood-soaked pledgets and transmission was tested using a capillary-tube method. The important Australian vectors, Aedes vigilax (Skuse) and Culex annulirostris Skuse, were used as internal controls for the alphaviruses and flaviviruses, respectively. Overall, Cx. gelidus was a more efficient vector of flaviviruses than alphaviruses. Culex gelidus was refractory to infection with BFV, and nearly 25% transmitted RRV, which was comparable to Ae. vigilax . Culex gelidus was susceptible to all three flaviviruses, with transmission rates of 96%, 95% and 41% for JEV, KUNV and MVEV, respectively. JEV transmission rates in Cx. annulirostris were unexpectedly low and this was possibly due to differences in susceptibility to JEV genotypes I and II. Considering the high susceptibility to the flaviviruses demonstrated here, and the natural infections with RRV and JEV that have been detected from northern Australian populations, the establishment of the exotic mosquito, Cx. gelidus , in Australia is potentially a significant public health concern.